1. Osteoclast formation induced by coculture Human osteoblast and monocyte cell lines

2. Coculture HOS+U937 Coculture HOS and U937 to induce osteoclast Exam design – Coculture in α-MEM medium – HOS 10^3 cell/well – U937 10^5 cell/well – Fixed by paraformaldehyde 10min at 2,4,6,8 days – Saved at PBS – Trap stain to check morphology culture2468

3. Hos 10 3 +U937 10 3 物鏡 20X 物鏡 4X 2day 6day4day

4. Hos 10 3 +U937 10 3 物鏡 20X 物鏡 4X

5. Indirect co-culture(Transwell) Exam design – Coculture in α-MEM medium – HOS 10^3 cell/well – U937 10^5 cell/well – Fixed by paraformaldehyde 10min at 8 days – Saved at PBS – Trap stain to check morphology HOS seeding in inner well 480 U937seeding in outer well U937HOS

6. Only little round cells attach to the floor, no giant cell is found at 4day

7. Indirect co-culture(condition medium) Exam design – Condition medium for induce OC HOS cell cultures 3 days past confluency in T75 flasks 40 % HOS medium + 60 % flash α-MEM medium with FBS – U937 10^5 cell/well – Fixed by paraformaldehyde 10min at 8 days and Saved at PBS – Trap stain to check morphology U937+condition medium 048

8. 4day controlCondition medium 物鏡 4X 物鏡 20X